pGEM-gbr22 Protein Expression, Purification, and Characterization
Introduction
When attempting to express a protein, E. Coli is the quickest, easiest and cheapest method of expression systems(1). E. coli is also the most obvious choice when expressing a protein of prokaryotic origin. E. Coli has all the machinery necessary for folding and post-translational modifications which proves to be very useful (1). However, many eukaryotic proteins don't fold properly in E. coli and form insoluble aggregates (inclusion bodies). In terms of full-length proteins, analysis of large-scale protein expression trials shows that up to 50% of proteins from the Eubacteria or Archaea and 10% of proteins from the Eukarya can be expressed in E. coli in soluble form (2). For Eukaryotic proteins it is important to use BL21(DE3) derivatives carrying additional tRNAs to overcome the effects of codon bias. There will also be post modifications needed for the protein to become active or adapt to the right structure. The simplest of these modifications is the removal of the N-terminal methionine residue, which can occur in all organisms(1). For purification there are many methods including columns or liquid chromatography. For column purification, the protein can be run through a column matrix which contains Ni-NTA resin buffer. The purification steps can be integrated by high-performance liquid chromatography which most commonly uses the ÄKTA systems from GE Healthcare(2). Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality.Characterization also includes UV absorption spectroscopy to quantify the amount and concentration of purified protein. It is also useful to measure the UV absorption at A280 and calculate the concentration of the protein by using the predicted molar extinction coefficient at A280. By taking a UV absorption spectrum, it is also possible to uncover contamination with DNA or RNA, or reveal common cofactors(2). Characterization include running the protein through a gel electrophoresis. By using gel electrophoresis, contaminations can be seen in the bands of protein that occur that is different from the pGEM-gbr22.
Materials and Methods Expression The E. Coli was added into two separate transformation tubes over an open flame. The pGEM-gbr22 plasmid was added into one of the transformation tubes. The transformation tube was heat shocked by placing it into ice followed by a heat bath of 42 degrees celcius and placed in an ice bath. SOC medua was placed in the tubes and the tubes were placed in a water bath incubator. The collirollers were placed in the plates to spread the bacteria and they were allowed to incubate overnight at 37 degrees celicus. A pipette tip was used to swipe a colony of transformed bacteria and was placed in a Erlenmeyer flask of LB and ampicillin. The flask was placed in a water bath shaker for 24 hours. The flask was poured into a 50 ml conical tube and placed in Allegra X-15 (Beckman Coulter, Inc., Brea, CA). The supernatant was decanted and the pellet was pipette up and down with a 2.5 ml of 11x PBS solution. The Lysozyme and Benzonase (Sigma-Aldrich, St. Louis, MO) was then added to the tube. Purification The tubes were put into three Eppendorf tubes and centrifuged. The supernatant was placed in a conical tube and then filtered through a PES syringe filter (Membrane Solutions, Plano, TX). An Econo column (Bio-Rad, Hercules, CA) was used to run the supernatant with Ni-NTA resin/buffer through. The column was first rinsed with nanopure water, then the supernatant with the Ni-NTA resin/buffer was ran through the column and then it was dripped into a 10 ml round bottom conical tube labeled 'waste'. 5 ml of the Wash solution was run through the column. 5 ml of the Elution solution was ran through the column in the same way as before. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, ED) was used to measure the absorbance of the protein from Elution 1 at 280 nanometers and 574 nanometers. Protein Characterization The protein was characterized by putting the 6X gel loading buffer into the pellet. The tubes were placed into a heat block and centrifuged at 5000 RPM for 2 minutes. The An SDS electrophoresis was run for twenty-five minutes at two hundred volts. The first well contained the protein ladder (Thermo Fisher Scientific, Waltham, MA) #SM0671, the following ladders contained samples one through six, and four through six from a partner.The gel was then stained. The next day the gel was placed on Whatman filter paper (GE Healthcare, Maidstone, UK) and covered with cellophane. The gel was then placed on a drying bed for 1.5 hours at 75 degrees Celsius on the Gradient cycle.
Results:
Figure 1: E. Coli BL21 colonies incubated at 37 degrees Celcius for 24 hours with ampicillin.
Figure 2: "Fun Plate", microorganisms collected belly button ring
Figure 3: E. Coli BL21 colonies that express a gene which is resistant to ampicillin. There were no colonies that could be seen since the cells were believed to have been lysed.
Figure 4: Image of E. Coli BL21 (DE3) bacteria with expressed pGEM-gbr22 protein resting in an Erlenmeyer Flask mixed with 25 ml LB broth and ampicillin media
Figure 5: Elution 1 which contains purified pGEM-gbr22 protein after washed down by Elution buffer solution in Ni-NTA column
Figure 6: Elution 2 which contains purified pGEM-gbr22 protein remaining after Elution 1 from the Ni-NTA column
Figure 7: pGEM-gbr22 purple protein after centrifuged using Allegra X-15 into a pellet form. The supernatant was removed from the conical tube and pellets weighs 0.24g
Figure 8: Nanodrop spectrophotometer absorbance spectra for Elution 1 sample. Beer’s Law is used (A=Ebc). At 280 nm, an extinction coefficient of 38,850was found , a pathlength of 1 cm, and an absorbance of 0.71. The concentration of 1.8 X 10-5 M was determined. Multiplying by the known molecular weight of the protein (25,794.2 g/mol), the concentration of protein was also determined to be 0.446 mg/ml.
Figure 9: Nanodrop spectrophotometer absorbance spectra for Elution 1 sample. Beer’s Law is used (A=Ebc). At 280 nm, an extinction coefficient of 38,850was found , a pathlength of 1 cm, and an absorbance of 0.70. The concentration of 1.78 X 10-5 M was determined. Multiplying by the known molecular weight of the protein (25,794.2 g/mol), the concentration of protein was also determined to be 0.452 mg/ml.
Figure 10: Nanodrop spectrophotometer absorbance spectra of first of two Elution 1 samples at 574 nm maximum pGEM-gbr22 protein wavelength. At 574 nm, we found an absorbance of .140, an extinction coefficient of 118,300 and a path length of 1 mm. The concentration was found to be 1.21 X 10-6 M . The concentration was then multiplied by the molecular weight of the protein (25,794.2 g/mol) making the concentration to be 0.0285 mg/ml.
Figure 11: Nanodrop spectrophotometer absorbance spectra of second of two Elution 1 samples at 574 nm maximum pGEM-gbr22 protein wavelength. At 574 nm, we found an absorbance of 0.128, an extinction coefficient of 118,300 and a path length of 1 mm. The concentration was found to be 1.08 X 10-6 M . The concentration was then multiplied by the molecular weight of the protein (25,794.2 g/mol) making the concentration to be 0.0279 mg/ml.
figure 12: Image of results of SDS gel electrophoresis ran for 25 minutes at 200 The column on the left represents a ladder from Fermentas. This is followed by Samples one through six collected throughout the experiment collected by partner, then samples four through six of original protein.
Figure 13: SDS gel electrophoresis ladder for fermantus
Discussion: The E. Coli bacteria that were ampicillin resistant were used to create the protein(pGEM-gbr22). Sample one consisted of the E. Coli, Amp and LB. The Sample two was created by centrifuging the E. Coli and using Lysozyme to break the cell wall and Benzonase to break down the DNA and RNA in the cell. The supernatant that resulted was the used for Sample two which was the soluble protein. Sample three was created by using the Ni-NTa resin to remove the histidine through the column. Sample three was the proteins without the histidine tag. Sample four used the wash which consisted of 20mM concentration of Imidazole and 1X PBS removing some proteins. Sample five and six used the elution which consisted of 250mM concentration of Imidazole and 1X PBS removing all of the proteins essentially. Sample 5 was the first elution and Sample 6 was the second elution. The last sample contained the remaining protein attached to the column. We used the Nanodrop Spectrophotometer for Elution 1 to see if there is any other proteins in our sample. Beer’s Law is used (A=Ebc). First two readings of the elution one was taken at 280 nm. At 280 nm, an extinction coefficient of 38,850was found , a pathlength of 1 cm, and an absorbance of 0.70 and 0.71. The concentration of 1.8 X 10-5 M was determined. Multiplying by the known molecular weight of the protein (25,794.2 g/mol), the concentration of protein was also determined to be 0.446 mg/ml. Another two readings were taken of the elution one at 574 nm. At 574 nm, we found an absorbance of 0.128 and .140, an extinction coefficient of 118,300 and a path length of 1 mm. On Average, the concentration was found to be 1.08 X 10-6 M . The concentration was then multiplied by the molecular weight of the protein (25,794.2 g/mol) making the concentration to be 0.0279 mg/ml. Using Gel electrophoresis, characterization was done to see if there was a major contamination in the protein. The sample 5 appears to have a slight band. There seems to be a small band in 6 and 7. There doesn’t seem to be many contaminations that occurred but the intensity of the band is not very good which means that there isn’t a great concentration of protein expressed. This was possibly a major source of error which could have occurred while adding a stronger concentration of Imidazole which washed away the protein in sample 4. There was also another source of error that occurred while attempting the expression of the protein in the E. Coli. The control plate had growth which means that ampicillin resistant bacteria was able to grow in the plate. The experimental plate also became unreliable since the colonies were not well defined which resulted from the accidental lysing of the cells. The lab was continued using a colony from another tube.
Conclusions: In this experiment we used E. Coli BL21 bacteria in order to express our protein of interest (pGEM-gbr22). The protein was purified by using Ni-NTA column along with wash and elution buffers to obtain the pGEM-gbr22 protein. There was however contaminations that occurred along the expression and purification process which could be seen quantitatively in the nanodrop and qualitatively in the gel electrophoresis. The use of bacteria to make protein is a crucial part of the process in Virtual Drug Screening. The characterization process is also necessary to ensure the proper protein is being created, This is crucial especially if the protein goes through enzyme assays. Creating the protein properly is necessary to test the ligands of the GOLD and PyMol program to test the binding ability.
References:
1) Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification.Nat Methods2008,5 (2), 135-46. 2) Zhang, S., Zubay, G. ; Goldman, E. Low-usage codons in Escherichia coli, yeast, fruit fly and primates. Gene 1991, 105 , 61-72.
3) Kane, J.F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli.Current Opinions Biotechnol.2001, 6, 494-500.
pGEM-gbr22 Protein Expression, Purification, and Characterization
Introduction
When attempting to express a protein, E. Coli is the quickest, easiest and cheapest method of expression systems(1). E. coli is also the most obvious choice when expressing a protein of prokaryotic origin. E. Coli has all the machinery necessary for folding and post-translational modifications which proves to be very useful (1). However, many eukaryotic proteins don't fold properly in E. coli and form insoluble aggregates (inclusion bodies). In terms of full-length proteins, analysis of large-scale protein expression trials shows that up to 50% of proteins from the Eubacteria or Archaea and 10% of proteins from the Eukarya can be expressed in E. coli in soluble form (2). For Eukaryotic proteins it is important to use BL21(DE3) derivatives carrying additional tRNAs to overcome the effects of codon bias. There will also be post modifications needed for the protein to become active or adapt to the right structure. The simplest of these modifications is the removal of the N-terminal methionine residue, which can occur in all organisms(1). For purification there are many methods including columns or liquid chromatography. For column purification, the protein can be run through a column matrix which contains Ni-NTA resin buffer. The purification steps can be integrated by high-performance liquid chromatography which most commonly uses the ÄKTA systems from GE Healthcare(2). Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality.Characterization also includes UV absorption spectroscopy to quantify the amount and concentration of purified protein. It is also useful to measure the UV absorption at A280 and calculate the concentration of the protein by using the predicted molar extinction coefficient at A280. By taking a UV absorption spectrum, it is also possible to uncover contamination with DNA or RNA, or reveal common cofactors(2). Characterization include running the protein through a gel electrophoresis. By using gel electrophoresis, contaminations can be seen in the bands of protein that occur that is different from the pGEM-gbr22.
Materials and Methods
Expression
The E. Coli was added into two separate transformation tubes over an open flame. The pGEM-gbr22 plasmid was added into one of the transformation tubes. The transformation tube was heat shocked by placing it into ice followed by a heat bath of 42 degrees celcius and placed in an ice bath. SOC medua was placed in the tubes and the tubes were placed in a water bath incubator. The collirollers were placed in the plates to spread the bacteria and they were allowed to incubate overnight at 37 degrees celicus. A pipette tip was used to swipe a colony of transformed bacteria and was placed in a Erlenmeyer flask of LB and ampicillin. The flask was placed in a water bath shaker for 24 hours. The flask was poured into a 50 ml conical tube and placed in Allegra X-15 (Beckman Coulter, Inc., Brea, CA). The supernatant was decanted and the pellet was pipette up and down with a 2.5 ml of 11x PBS solution. The Lysozyme and Benzonase (Sigma-Aldrich, St. Louis, MO) was then added to the tube.
Purification
The tubes were put into three Eppendorf tubes and centrifuged. The supernatant was placed in a conical tube and then filtered through a PES syringe filter (Membrane Solutions, Plano, TX). An Econo column (Bio-Rad, Hercules, CA) was used to run the supernatant with Ni-NTA resin/buffer through. The column was first rinsed with nanopure water, then the supernatant with the Ni-NTA resin/buffer was ran through the column and then it was dripped into a 10 ml round bottom conical tube labeled 'waste'. 5 ml of the Wash solution was run through the column. 5 ml of the Elution solution was ran through the column in the same way as before. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, ED) was used to measure the absorbance of the protein from Elution 1 at 280 nanometers and 574 nanometers.
Protein Characterization
The protein was characterized by putting the 6X gel loading buffer into the pellet. The tubes were placed into a heat block and centrifuged at 5000 RPM for 2 minutes. The An SDS electrophoresis was run for twenty-five minutes at two hundred volts. The first well contained the protein ladder (Thermo Fisher Scientific, Waltham, MA) #SM0671, the following ladders contained samples one through six, and four through six from a partner.The gel was then stained. The next day the gel was placed on Whatman filter paper (GE Healthcare, Maidstone, UK) and covered with cellophane. The gel was then placed on a drying bed for 1.5 hours at 75 degrees Celsius on the Gradient cycle.
Results:
Discussion:
The E. Coli bacteria that were ampicillin resistant were used to create the protein(pGEM-gbr22). Sample one consisted of the E. Coli, Amp and LB. The Sample two was created by centrifuging the E. Coli and using Lysozyme to break the cell wall and Benzonase to break down the DNA and RNA in the cell. The supernatant that resulted was the used for Sample two which was the soluble protein. Sample three was created by using the Ni-NTa resin to remove the histidine through the column. Sample three was the proteins without the histidine tag. Sample four used the wash which consisted of 20mM concentration of Imidazole and 1X PBS removing some proteins. Sample five and six used the elution which consisted of 250mM concentration of Imidazole and 1X PBS removing all of the proteins essentially. Sample 5 was the first elution and Sample 6 was the second elution. The last sample contained the remaining protein attached to the column. We used the Nanodrop Spectrophotometer for Elution 1 to see if there is any other proteins in our sample. Beer’s Law is used (A=Ebc). First two readings of the elution one was taken at 280 nm. At 280 nm, an extinction coefficient of 38,850was found , a pathlength of 1 cm, and an absorbance of 0.70 and 0.71. The concentration of 1.8 X 10-5 M was determined. Multiplying by the known molecular weight of the protein (25,794.2 g/mol), the concentration of protein was also determined to be 0.446 mg/ml. Another two readings were taken of the elution one at 574 nm. At 574 nm, we found an absorbance of 0.128 and .140, an extinction coefficient of 118,300 and a path length of 1 mm. On Average, the concentration was found to be 1.08 X 10-6 M . The concentration was then multiplied by the molecular weight of the protein (25,794.2 g/mol) making the concentration to be 0.0279 mg/ml. Using Gel electrophoresis, characterization was done to see if there was a major contamination in the protein. The sample 5 appears to have a slight band. There seems to be a small band in 6 and 7. There doesn’t seem to be many contaminations that occurred but the intensity of the band is not very good which means that there isn’t a great concentration of protein expressed. This was possibly a major source of error which could have occurred while adding a stronger concentration of Imidazole which washed away the protein in sample 4. There was also another source of error that occurred while attempting the expression of the protein in the E. Coli. The control plate had growth which means that ampicillin resistant bacteria was able to grow in the plate. The experimental plate also became unreliable since the colonies were not well defined which resulted from the accidental lysing of the cells. The lab was continued using a colony from another tube.
Conclusions:
In this experiment we used E. Coli BL21 bacteria in order to express our protein of interest (pGEM-gbr22). The protein was purified by using Ni-NTA column along with wash and elution buffers to obtain the pGEM-gbr22 protein. There was however contaminations that occurred along the expression and purification process which could be seen quantitatively in the nanodrop and qualitatively in the gel electrophoresis. The use of bacteria to make protein is a crucial part of the process in Virtual Drug Screening. The characterization process is also necessary to ensure the proper protein is being created, This is crucial especially if the protein goes through enzyme assays. Creating the protein properly is necessary to test the ligands of the GOLD and PyMol program to test the binding ability.
References:
1) Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification.Nat Methods2008,5 (2), 135-46.
2) Zhang, S., Zubay, G. ; Goldman, E. Low-usage codons in Escherichia coli, yeast, fruit fly and primates. Gene 1991, 105 , 61-72.
3) Kane, J.F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Current Opinions Biotechnol. 2001, 6, 494-500.